Study On The RSL1D1-p53 Interaction And Preparation Of Anti-RSL1D1 Single-Chain Variable Fragment

The protein encoded by Ribosomal L1-domain-containing(RSL1D1),also known as CSIG,belongs to the ribosomal L1p/L10e family.It was first discovered and cloned when the human embryo lung diploid fibroblasts were screened by suppression subtractive hybridization during senescence.Recent studies have shown that RSL1D1 plays an important role in tumor cell proliferation and apoptosis by regulating the p53-MDM2 signaling pathway and is a crucial new target in tumor research.Malignant tumor is a kind of serious threat to human health.At present,engineering antibody has become one of the most important tools for tumor treatment.As a representative of small-molecule antibodies,a single-chain variable fragment(ScFv)has become a hot spot in the field of antibody engineering due to its advantages of reduced immunogenicity,small molecular weight and short retention time in vivo.This study aims to elucidate the mechanism RSL1D1-p53 interaction and construct an anti-RSLIDI ScFv to provide theoretical and material basis for anti-tumor therapy targeting RSL1D1.First,the domains mediated RSL1D1-p53 interaction was identified by GST pulldown in vitro.Previously,our research team has found that RSL1D1 can physically interact with p53 and regulate the p53 signaling pathway.To further reveal the mechanism involved,we constructed a series of truncated mutants of RSL1D1 and p53 according to the published protein structures.After obtaining purified proteins through prokaryotic expression and affinity chromatography,we carried out a panel of GST pulldown assays and found that both the N and C termini of RSL1D1 interacted with the central DNA binding domain of p53.Then,we prepared anti-RSL1D1 monoclonal antibodies by a routine technique.We immunized female Balb/c mice with purified truncated RSL1D1 mutant proteins,fused spleen cells with SP2/0 myeloma cells,screened and monoclonalized the fusion cells,and obtained 27 strains of anti-RSL1D1 hybridoma cells.Three out of 27 strains(1E5-1E10,1C9-2C8,1D8-2B10)were chosen for production of ascites,followed by purification of monoclonal antibodies via Protein A affinity chromatography.The subtypes,purity,molecular weight,titer,sensitivity,affinity constant and specificity of the three antibodies were detected by SDS-PAGE,westerm blot and indirect ELISA;1E5-1E10 is high sensitivity,affinity and specificity with the subtypy of IgG1,a purity of 95%or more,titration of 1:10000,detection limit of 0.1 ng,affinity constant of(4.85±0.48)×108 L/mol;The endogenous RSL1D1 protein is detected as a specific single band.Finally,an anti-RSLIDI ScFv was constructed and identified.Total RNA was extracted from the identified 1E5-1E10 hybridoma,and cDNA was synthesized by reverse transcription.Genes encoding the heavy and light chain variable regions of antibodies were amplified by PCR,and the ScFv was constructed by linking the variable regions of heavy and light chains with a flexible peptide linker.The nucleotide sequence of the ScFv gene was inserted into a prokaryotic expression vector pET-32a-SUMO,and the ScFv fusion protein was induced in Escherichia coli BL21(DE3)and purified by affinity chromatography.The interaction between the ScFv and RSL1D1 was verified by GST pulldown and bimolecular fluorescent complementation assays.The results show that ScFv in this study can direct bind to the N-terminus of RSL1D1.In summary,this study proved that RSL1D1 can directly bind to p535 and both its N and C terminal domains are directly bound to the central DNA binding domain of p53(102-292 amino acids).We speculate that RSL1D1 inhibits the activity of p53 by directly binding to p53,which provides an important clue for tumor treatment targeting RSL1D1.In addition,an anti-RSLIDI ScFv has been successfully constructed in this study,providing a material basis for subsequent demonstration and research.